- electrophoresis problems, e.i. quality of gel or Laemli sample loading buffer (better to use a fresh one)
- if the first problem is excluded, you may think about proteolysis. Thus, you have to use protease inhibitors and perform WB as soon as possible after sample preparation.
- post-translational modification(s) of you protein. If so, it has to be studied in details and can be a big peace of work.